Effects of matcha tea extract on cell viability and estrogen receptor-β expression on MCF-7 breast cancer cells

MCF-7 cells were chosen for their hormone receptor-positive attributes, reflecting the luminal breast cancer subtype. Their estrogen and progesterone receptor presence aligns with our hormone-related focus. Our selection was guided by their relevance to our objectives, well-documented status, and specific interest in MTE's impact on this hormone receptor-positive subtype. Additionally, our choice of MCF-7 cells was influenced by the existing data on green tea phenol's interaction with ERα, while information about ERβ interactions remains limited.

MCF-7 cells were cultured on an 80% monolayer in a cell culture bottle. Dulbecco's modified Eagle medium (DMEM; 3.7 g/l NaHCO3, 4.5 g/l d-glucose, 1.028 g/l stable glutamine, and sodium pyruvate; Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS; Biochrom, Berlin, Germany) was used for cultivation. The cells were incubated with atmospheric concentrations of CO₂ of 5% at 37 °C and were then trypsinized and counted for further use.

The matcha tea extract was commercially purchased (Houjo Matcha Tea, harvested in Hoshino, Yame prefecture, Japan). MTE was prepared as described in the individual tests below.

MCF-7 cells were cultured in a 96-well plate at a density of 10,000 cells per well in 50 µl of DMEM with 10% FCS. After 4 h, the medium was changed to DMEM without FCS. FCS can contain substances such as growth factors, hormones, vitamins, and transport proteins, which can potentially influence cell proliferation and maintenance. The use of DMEM without FCS helps to reduce this influence on the experimental setup [12, 13]. The cells were further incubated for 12 h.

MTE (27.1 mg) was dissolved in 100 µl of pure ethanol and then diluted 1:1000 with DMEM without FCS. The solution for the control group was diluted in the same manner without adding MTE. Next, different quantities of MTE solution and DMEM without FCS were added to achieve different concentrations (5 µg/ml and 10 µg/ml). A total of 100 µl was added per well. For the control groups, the control solution and DMEM without FCS were pipetted into each well instead of the MTE solution. Incubation was carried out for 72 h.

Cell incubation was conducted in a 12-well plate with a density of 500,000 cells per well for 4 h using 500 µl of DMEM with 10% FCS. Subsequently, the medium was changed to 500 µl of DMEM without FCS, and the MCF-7 cells were further incubated for 12 h. For the preparation of MTE solution, 27.1 mg of MTE was dissolved in 100 µl of pure ethanol, and then diluted at a ratio of 1:1000 with DMEM without FCS. The solution for the control group was prepared in the same manner without adding MTE.

To obtain different concentrations (5 µg/ml and 10 µg/ml), various amounts of MTE solution and DMEM without FCS were added, totaling 500 µl per well. In the control group, the control solution and DMEM without FCS were added to each well instead of the MTE solution. This was followed by a 2-h incubation period. Since changes in mRNA levels can occur within hours after stimulation, and the half-life of mRNA is also in the range of hours, a 2-h incubation time was chosen. After incubation, excess liquid was removed, and the wells were rinsed with phosphate-buffered saline (PBS). RA-1 buffer (Macherey–Nagel, Düren, Germany) was added to lyse the cells.

RNA isolation was performed using NucleoSpinRNAII (Macherey–Nagel, Düren, Germany), and reverse transcription of the RNA was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) with 10 ng of RNA. The temperature protocol included phases of 10 min at 25 °C, 2 h at 37 °C, 5 s at 85 °C, followed by cooling to 4 °C.

For the PCR assay, each well was prepared with 10 µl of TaqMan^® Universal PCR Master Mix 2X (Thermo Fisher Scientific), 8 µl of distilled water (treated with 0.1% diethyl pyrocarbonate), 1 µl of TaqMan^® Gene Expression Assay 20X (Thermo Fisher Scientific; Target: ACTB, Assay ID Hs999903_m1; Target: ESR2, Assay ID Hs01100357_m1; primer sequences not provided by the manufacturer), and 1 µl of cDNA sample. The PCR assay was performed using the ABI Prism 7500 Fast (Thermo Fisher Scientific).

Thermal cycling for the PCR assay was conducted as follows: the reaction started with an initial denaturation step at 95 °C for 20 s, followed by 40 cycles of amplification at 95 °C for 3 s, and then 60 °C for 30 s. The results were analyzed using the comparative 2−ΔΔCT method [14], with β-actin serving as the endogenous control for the calculation of ΔCT values. As previously implemented in the WST-1 assay, a total of three measurements were conducted, each accompanied by two technical replicates. For RNA isolation, NucleoSpinRNAII kit (Macherey–Nagel, Düren, Germany) was used, and reverse transcription of the RNA was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) with 10 ng of RNA. The temperature protocol included phases of 10 min at 25 °C, 2 h at 37 °C, 5 s at 85 °C, followed by cooling to 4 °C.

MCF-7 cells were cultured in 1000 µl of DMEM with 10% FCS in a 12-well plate at a density of 500,000 cells per well. After 4 h, the medium was changed to 1000 µl of DMEM without FCS, and the cells were incubated for an additional 12 h. To prepare the MTE (20 mg) solution, it was dissolved in 100 µl of pure ethanol and then diluted in a ratio of 1:1000 with DMEM without FCS. The control group was prepared in the same way without adding MTE. Test groups with desired concentrations of 5 µg/ml and 10 µg/ml were achieved by pipetting different amounts of the MTE solution and DMEM without FCS, resulting in a total volume of 1000 µl per well. For the control group, 250 µl of control solution and 750 µl of DMEM without FCS were added to each well instead of the MTE solution. The cells were then cultured for 48 h and rinsed with phosphate-buffered saline (PBS). To lyse the cells, 200 µl of a buffer solution containing a 1:100 dilution of protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) in RIPA buffer (radioimmunoprecipitation assay buffer; Sigma-Aldrich) was added to each well. The cells were then incubated for 30 min at 4 °C and centrifuged to obtain the supernatant for Bradford protein assay. The proteins were separated by SDS-PAGE based on their molecular weight and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). The PVDF membrane was blocked for 1 h with a 1 × casein solution (Vector Laboratories, Burlingame, CA, USA) to prevent nonspecific binding of antibodies. The primary antibodies used as endogenous controls were anti-β-actin (clone AC-15, mouse IgG; Sigma-Aldrich Co., St. Louis, Missouri, USA) and anti-ERß (polyclonal IgG, rabbit, Abcam, Cambridge, UK), which were diluted in a 1 × casein solution and incubated on the membrane for 16 h at 4 °C. After rinsing the membranes with Tris-buffered saline (TBST), the membranes were incubated with biotinylated anti-rabbit IgG antibody and ABC-AmP reagent (VECTASTAIN ABC-AmP Kit for rabbit IgG, Vector Laboratories) according to the manufacturer's protocol. The specific bands on the membrane were visualized using the BCIP/NBT chromogenic substrate (Vectastain ABC-AmP Kit, Vector Laboratories) and detected with the Bio-Rad Universal Hood II (Bio-Rad Laboratories, Hercules, CA, USA). The intensity of the color in the ERβ bands was quantified by comparing the clustered pixels to the β-actin bands using the Bio-Rad Quantity One software (Bio-Rad Laboratories). An example of a western blot is shown in Fig. 1. The western blots were repeated nine times for statistical analysis.

The statistical programming environment R, version 4.0.2 [15], was utilized for data processing and statistical analysis. p values less than 0.05 were considered statistically significant. Normality of distribution was assessed using Shapiro–Wilk tests. Due to the distinct experimental designs, different tests were employed to compare the experimental groups and control groups. For the WST-1 and PCR assay, paired t tests were used. For the western blot analyses, a single-factor ANOVA with post hoc test was employed.

A WST-1 proliferation assay was conducted to assess the impact of incubation on MCF-7 cellular viability. Statistical significance was observed only at higher concentrations of MTE, with p values of 0.074 at 5 µg/ml MTE and 0.017 at 10 µg/ml MTE. The results are presented in Fig. 2.

The MTE TaqMan^® PCR was used to detect changes in ERß mRNA expression in MCF-7 cells after incubation with MTE. The comparative 2−ΔΔCq method was employed for data analysis. However, there were no significant changes observed in the x-fold expression of mRNA in comparison to the control, for both MTE concentrations (5 µg/ml MTE: p = 0.091 and 10 µg/ml MTE: p = 0.838). The results are depicted in Fig. 3.

The western blot technique was employed to examine alterations in ERß expression at the protein level. Remarkably, a noteworthy decrease in ERß expression was observed solely in the higher concentrations of MTE (5 µg/ml MTE: p = 0.233 and 10 µg/ml MTE: p = 0.036), as illustrated in Fig. 4.